Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.
A number of macrocyclic peptides with diverse biological activities have been discovered in plants in the Rubiaceae and Violaceae families. These include kalata B1 (1), the circulins (2), cyclopsychotride (3) and several peptides from Viola species (4-6). They range in size from 29-31 amino acids and contain six conserved Cys residues. These macrocyclic peptides differ from classical proteins in that they have no free N- or C-terminus due to their amide-circularized backbone. They also incorporate a cystine knot in which an embedded ring in the structure formed by two disulfide bonds and their connecting backbone segments is threaded by a third disulfide bond. These combined features of the cyclic cystine knot (CCK) produce a unique protein fold that is topologically complex and has exceptional chemical and biological stability.
Small cyclic peptides are also known in nature, particularly as antibiotics of microbial origin, and appear to have advantages of improved stability and biological activity over their non-cyclic counterparts. Because of their favourable properties, cyclic peptides (or mimics of them) have had pharmaceutical applications. One example is the immunosuppressant, cyclosporine. These classical cyclic peptides invariably comprise fewer than 15 amino acids, usually lack disulfide bonds and generally do not have well defined three dimensional structures. Such peptides are not gene products but are thought to be biosynthesized, non-ribosomally, via peptide synthetases.
In work leading up to the present invention, the inventors investigated the genetic basis of the macrocyclic peptides. In contrast to small cyclic peptides, the macrocyclic peptides, referred to herein as “cyclotides”, are encoded for by gene sequences and exhibit folding structures characteristic of true proteins. The elucidation of the genetic basis behind the cyclotides enables their expression and manipulation in transgenic plant, animal and microbial cells. Being cyclic, the cyclotides have a range of potential therapeutic, diagnostic, industrial and agricultural including horticultural applications. The cyclizing enzyme or enzymes themselves also have utility in the development of in vivo or in vitro systems for cyclizing target peptides, polypeptides and proteins. Furthermore, the present invention permits the generation of linear structural homologues of peptides, polypeptides and proteins.